The goal of this study was to assess the mechanisms by which the caffeine-sensitive calcium stores of airway smooth muscle cells are refilled. Bovine trachealis cells were loaded with fura 2-AM (0.5 µM) for imaging of cytosolic calcium concentrations ([Ca²⁺]_i) in the inner cytosol. After a first stimulation (S1) with caffeine, the response to a second stimulation (S2) depended on the presence of extracellular calcium during an intervening 80-s-long refilling phase. The S2-to-S1 ratio (S2/S1) was 0.11 ± 0.05 (n = 13 cells) during calcium-free refilling but 0.72 ± 0.04 (n = 36 cells) within 80 s of exposure to extracellular calcium. Maximum mean [Ca²⁺]_i during the 80 s of refilling was not different for calcium-free (116 ± 19 nM; n = 13 cells) versus extracellular calcium plus nickel (2 mM) (121 ± 12 nM; n = 21 cells); despite this, significantly greater refilling (S2/S1 0.58 ± 0.06; n = 24 cells) occurred in the presence of extracellular calcium plus nickel. The protein tyrosine kinase inhibitors genistein (100 µM) and ST-638 (50 µM) significantly decreased refilling over 80 s (S2/S1 0.35 ± 0.06, n = 14 cells and 0.51 ± 0.07, n = 14 cells, respectively). Daidzein (100 µM) had no effect on S2/S1. We concluded that [Ca²⁺]_i of the inner cytosol during refilling correlated poorly with S2/S1 values and that, therefore, additional compartments not well detected by fura 2 contribute to refilling. The findings suggest that calcium influx for refilling is segregated from the inner cytosol of the cell, relatively insensitive to nickel, and regulated or modulated by protein tyrosine kinase activity.